J A Renye1, G A Somkuti. 1. Eastern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Wyndmoor, PA 19038, USA. john.renye@ars.usda.gov
Abstract
AIMS: To test whether a single vector, nisin-controlled expression (NICE) system could be used to regulate expression of the pediocin operon in Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactobacillus casei. METHODS AND RESULTS: The intact pediocin operon was cloned immediately into pMSP3535 downstream of the nisA promoter (PnisA). The resulting vector, pRSNPed, was electrotransformed into Strep. thermophilus ST128, L. lactis subsp. lactis ML3 and Lact. casei C2. Presence of the intact vector was confirmed by PCR, resulting in the amplification of a 0.8-kb DNA fragment, and inhibition zones were observed for all lactic acid bacteria (LAB) transformants following induction with 50 ng ml(-1) nisin, when Listeria monocytogenes Scott A was used as the target bacterium. Using L. monocytogenes NR30 as target, the L. lactis transformants produced hazy zones of inhibition, while the Lact. casei transformants produced clear zones of inhibition. Zones of inhibition were not observed when the Strep. thermophilus transformants were tested against NR30. CONCLUSIONS: The LAB hosts were able to produce enough pediocin to inhibit the growth of L. monocytogenes Scott A; the growth of L. monocytogenes NR30 was effectively inhibited only by the Lact. casei transformants. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the NICE system has been used to express the intact pediocin operon in these LAB hosts. This system could allow for the in situ production of pediocin in fermented dairy foods supplemented with nisin to prevent listeria contamination.
AIMS: To test whether a single vector, nisin-controlled expression (NICE) system could be used to regulate expression of the pediocin operon in Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactobacillus casei. METHODS AND RESULTS: The intact pediocin operon was cloned immediately into pMSP3535 downstream of the nisA promoter (PnisA). The resulting vector, pRSNPed, was electrotransformed into Strep. thermophilus ST128, L. lactis subsp. lactis ML3 and Lact. casei C2. Presence of the intact vector was confirmed by PCR, resulting in the amplification of a 0.8-kb DNA fragment, and inhibition zones were observed for all lactic acid bacteria (LAB) transformants following induction with 50 ng ml(-1) nisin, when Listeria monocytogenes Scott A was used as the target bacterium. Using L. monocytogenes NR30 as target, the L. lactis transformants produced hazy zones of inhibition, while the Lact. casei transformants produced clear zones of inhibition. Zones of inhibition were not observed when the Strep. thermophilus transformants were tested against NR30. CONCLUSIONS: The LAB hosts were able to produce enough pediocin to inhibit the growth of L. monocytogenes Scott A; the growth of L. monocytogenes NR30 was effectively inhibited only by the Lact. casei transformants. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the NICE system has been used to express the intact pediocin operon in these LAB hosts. This system could allow for the in situ production of pediocin in fermented dairy foods supplemented with nisin to prevent listeria contamination.
Authors: Ana Solopova; Cécile Formosa-Dague; Pascal Courtin; Sylviane Furlan; Patrick Veiga; Christine Péchoux; Julija Armalyte; Mikas Sadauskas; Jan Kok; Pascal Hols; Yves F Dufrêne; Oscar P Kuipers; Marie-Pierre Chapot-Chartier; Saulius Kulakauskas Journal: J Biol Chem Date: 2016-03-28 Impact factor: 5.157